The present invention relates to the field of chromatography and more particularly toward an apparatus and method for concentrating an analyte prior to an analytical detector such as a mass spectrometer.
Although analytical instrumentation is becoming increasingly sensitive and analyte detection continues to improve, many chemical analytes require concentration prior to chemical analysis. Typically this is done using bench-top chemical processes specifically developed or tailored to the analytical problem. Representative of these approaches are solvent condensation or evaporation techniques that eliminate the solvent while retaining the analyte (or analytesxe2x80x94in all cases analyte could just as well as be analytes) by exploiting differences in physical properties such as volatility. Nitrogen or inert gas xe2x80x9cblown-downsxe2x80x9d, rotary evaporation, Kuderna-Danish condensers, distillation (steam, etc.), tube heaters, vacuum evaporation, (freeze drying), and related techniques are typical of approaches to pre-concentrate an analyte by removal of a solvent. A number of commercial machines exist specifically for this purpose such as RapidVap(trademark), CentriVap(trademark), SafetyVap(trademark), as examples. Physical techniques such as centrifugation or selective adsorption (solid-phase extraction, column chromatography), selectively separate analytes from a bulk phase prior to redesolving in another solvent prior to injection. It is after these bench-chemistry steps that an analyte has been concentrated to an appropriate degree that makes detection and quantitation possible with an existing detector. These steps are time-consuming, subject to loss, usually require specialized equipment and technical expertise all of which lead to increased expense.
In gas chromatography, large volume injection techniques have been developed with special hardware (pre-column inlets) to allow more sensitive detection by evaporation of solvent while attempting to retain analyte inside the pre-column inlets prior to the analyte being delivered to the analytical column for chromatographic separation and analysis/detection. Typical volumes are less than or equal to 100-xcexcl (by single injection) and the most frequent approach is to inject the solution, either in portion or in entirety, then evaporate the solvent, which is vented from the chromatograph, and transfer the analyte to the analytical column. This approach suffers from a limitation on volume that can be contained inside the pre-column inlet and, therefore, any increases in volume must be obtained by consecutive injections and evaporation cycles that can result in sample losses. These pre-column inlets that thermally program the vaporization of the solvent are called PTVs.
Another similar approach is the cool-on-column solvent venting arrangements that use a long, large diameter of capillary tubing (approximately 1 ml volume) to retain the injected volume. The operation is again the same as the PTV in that the temperature is programmed to vaporize the solvent and retain the analyte. Again the injection volume is fixed by the mechanical configuration of the assorted tubing. In both these approaches the ability to retain analytes is determined by the difference in the boiling point between the solvent and the analyte and the ability of the pre-column inlet and /or analytical column (phase) to selectively capture and retain the analyte.
However, these large-volume pre-column inlets have been considered attractive relative to the standard pre-column inlet such as split/splitless or on-column that only allow less than 4-xcexcl injections and provide no capability for in-situ concentration.
Both the standard volume pre-column inlet arrangements and the existing large volume port technologies are limited in the volume that can be concentrated by the fact that liquid injections vaporize inside the port and mechanical arrangements (namely, the liquid or vapor volume that can be contained) place an upper bound on the concentration factors that can be achieved. It would be desirable to obtain in-situ concentration that is flexible and less constrained in the concentration factors that can be obtained.
In the biochemical and organic chemistry fields there is often a need to concentrate an analyte before it is loaded into an analytical instrument. For instance, some products may need to be concentrated, because they are expensive to derivatize, difficult to extract or synthesize. Small amounts of product or intermediates are produced and need to be characterized and identified accurately before proceeding to future research steps. However, low concentrations of product can be a problem for a researcher, because they may challenge the limits of instrument sensitivities or increase the possibility of inaccurate abundance measurements. In addition, the transfer of these analytes from instrument to instrument or from instrument to storage container can result in significant additional loss of product. For these reasons, simple techniques and instruments for both concentrating and analyzing analytes would be of interest to researchers.
A number of instruments already exist for concentrating analytes. For instance, in the biochemical fields analytes may be concentrated using centrifuges, ultra-centrifuges and filtering. A number of commercial products exist for this purpose. For instance, Amicon(trademark) produces a number of filters that allow for desalting of samples, removal of solvent and concentration of small amounts of analyte. However, these devices often require access to a centrifuge or micro-centrifuge for spinning the analytes and are effective only when concentrating small volumes.
On a larger scale, typical analyte concentration is performed on the bench top using rotary evaporation, K-D, dry nitrogen blow down or in a PTV injection port by depositing the analyte in a liner and then evaporating the solvent by heating the liner. Each of these methods provides for concentration of analyte by removal of solvent. However, most of these methods and instruments suffer from the limitation of possible loss of analyte. In addition, since the concentration of analyte is performed separately, these methods can be time intensive and laborious. It would, therefore, be of particular interest to be able to concentrate an analyte without having to transfer, or resolvate the analyte.
Recent trends in analytical instrumentation include components for concentrating analytes in situ (i.e. directly in the instrument or more preferably before the analytes are introduced onto the pre-column). For instance, quartenary pumps are being used in HPLC instruments to pre-mix analytes before the analyte is injected and separated. Present chromatographic technology, however, requires additional hardware, components or complex instrumentation design to appropriately concentrate analytes. In addition, most of the chromatography instrumentation is designed to concentrate the analyte inside the pre-column just before it is introduced onto the column. A problem with this type of device is that the analyte is too dilute or contains a volatile solvent that significantly lowers the amount of sample concentration. It would, therefore, be desirable to be able to adapt existing chromatography hardware to serve the purpose of concentrating analytes in situ (i.e. on the syringe needle tip). A brief review of pre-column inlets, therefore, is in order to clarify existing technology that may be adapted to serve these purposes.
A number of different pre-column inlets or injectors exist to provide accurate, reproducible and predictable introduction of analytes into gas chromatography columns. Usually the analyte is a liquid and can be injected using a syringe, but other devices are available. For instance, analytes can be introduced onto columns by automatic analyzers or valves. Pre-column inlets can be divided into two major categories including packed-column inlets and capillary-column inlets. In gas chromatography the packed column inlets are fairly popular. A second type of column called a capillary-column inlet is also quite popular. These inlets include: capillary direct or (vaporizing inlet), split/splitless inlets (a vaporizing inlet), programmed temperature vaporizer inlets (vaporizing) and cool-on-column (non-vaporizing) inlets.
Capillary inlets are used with wide-bore capillary columns (I.D.xe2x89xa70.5 mm) and are made by substituting a special insert inside a packed-column inlet. The two types of inlets, including the split and splitless inlets, are quite different in design and operation. The split inlet was the earlier design and is a vaporizing inlet that vents most of the analyte in the split mode and transfers most of it to the column in the splitless mode. The splitless inlets load the analyte directly onto the column whereas the split inlets allow for loading partial amounts of analyte onto the column while at the same time allows venting of a portion of the final analyte. Programmed temperature vaporizer (PTV) inlets combine the benefits of split, splitless and on-column inlets. Analytes are usually injected into a cool liner and no syringe needle discrimination can occur. Inlet temperature is then usually increased to vaporize the analyte. A variety of user programs can then be used to provide determined vent times and temperatures to achieve results similar to split or spiitless transfers. PTV injection provides the most flexibility. Other advantages of PTV inlets include: the ability to trap nonvolatile components in a liner, removal of solvent and low boiling components, use of large injection volumes, no special syringe needed, no syringe needle discrimination, retention time and high reproducibilities similar to cool on column injections. A number of commercial injectors exist for these purposes. For instance, the Apex Prosep(trademark) pre-column inlet is highly versatile and may be used for injecting analytes. The system includes a standard inlet that contains both temperature and vaporizing regulation. However, these systems have only been used to concentrate analytes after they have been ejected from the syringe needle into the pre-column inlet. A number of techniques are used at this point to concentrate, or focus, the injected analtye. The major focusing techniques include stationary phase focusing, solvent focusing and thermal focusing.
Stationary phase focusing is the most frequently used technique and is possible only in temperature-programmed analysis. Generally, in gas chromatography, retention is a function of temperature. For instance, the speed at which the solutes travel down the column is dependent upon the temperature changes. As the temperature is increased, the solutes increase their speed of separation and as the temperature is lowered, they slow their movement. As the vaporizing analyte moves from the inlet to the column, it comes in contact with the stationary phase and is trapped in a defined range. The lower the applied temperature, the more effective the focusing.
Solvent focusing occurs in a different fashion compared to stationary phase focusing. For instance, as a condensed solvent starts to evaporate, solutes with volatility similar to that of the solvent tend to concentrate and focus on the solvent tail. This technique yields very narrow peaks.
Thermal focusing is the last technique and relates to the condensation of gases in a tube or at the head of the column. Peaks narrow as the solute volume is reduced during condensation. This technique is particularly helpful in narrowing bandwidths when the column temperature is approximately 150xc2x0 C. below the boiling point of the solvent. In addition, thermal focusing does not rely on chromatographic processes. It only requires a surface on which vapors can condense. To date, each of these techniques has been applied to a gas chromatography column or pre-column inlets. These techniques are not designed for concentrating the analyte at the pre-column stage. A disadvantage of this is that lower amounts of analyte are loaded onto the column and by the time the separated components reach the final mass analyzer, a low signal to noise ratio is obtained. This negatively affects the overall results. Therefore, there is a substantial need for an apparatus and method that concentrates the analyte before it is injected into the pre-column inlet.
Accordingly, it is an object of the present invention to overcome the above-mentioned disadvantages of the prior art by providing a novel method and apparatus for selectively removing solvent and concentrating an analyte before it is subject to chromatographic analysis.
It is another object of the invention to provide an improved method and apparatus for concentrating an analyte in situ using an instrument pre-column inlet port and syringe.
It is a still further object of the invention to provide an improved method of removing solvent from an analyte before injection into a pre-column inlet port or chromatographic column.
Additional objects, advantages and novel features of the invention set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by the practice of the invention.
In a general aspect, then, the present invention relates to an apparatus for concentrating an analyte at a gas chromatograph (GC). The apparatus has a dispensing means for holding and dispensing an analyte, an injection means for receiving the dispensing means, and a concentration means in contact with the injection means for concentrating the analyte on the dispensing means. The dispensing means may include an auto-sampler having a syringe with a needle and tip or similar type device. The dispensing means may also comprise the syringe needle and tip without the auto-sampler. The injection means includes a pre-column inlet having an inlet port designed for receiving the dispensing means. The concentration means is in contact with the injection means and is designed for evaporating or heating and removing the solvent from the analyte at the dispensing means. The concentration means may be a heater or a gas inlet port.
The invention also includes a method of removing solvent from an analyte and concentrating the analyte in a mass spectrometer. The technique accomplishes the concentration before the analyte is injected into a pre-column inlet and loaded onto a gas chromatograph by first collecting the analyte in the dispensing means. The dispensing means is then inserted into the injection means and the solvent is removed from the analyte through evaporation. The evaporation may occur by heating the analyte or by applying a gas stream. The concentrating means uses a combination of solvent and/or thermal focusing to concentrate the analyte.
Additional objects, advantages and novel features of the invention will be set forth in the part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.